Genetic recombination is a fundamental biological process that occurs in virtually all organisms. Site-specific recombination is a class of recombination events that are mediated by specialized enzymes acting at specific DNA sites, and catalyze a broad array of biological outcomes includes viral integration, bacterial antigen switching, and generation of immunological diversity. The goal of this proposal is to understand the mechanism and regulation of site-specific recombination. Our model system for investigation is the integration and excision of mycobacteriophage Bxb1, a virus that infects mycobacterial hosts. These host bacteria are of considerable medical importance, and include Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of human tuberculosis and leprosy respectively. The genetic systems required for their manipulation remains rudimentary and mycobacteriophage characterization has great potential for contributing to the development of improved vaccines, new drugs, and speedier diagnostic tools. The integration and excision system of mycobacteriophage Bxb1 is unusual in that these recombination events are catalyzed by an integrase protein that is a member of the family of serine-recombinases. These recombinases were identified relatively recently, and preliminary analysis shows that their mechanism is distinctly different to the well-characterized tyrosine-recombinase integrases. More specifically, the Bxb1 integrase acts to recombine two DNA sites - attB and attP - that are small (<50bp), different in sequence and size, and requires no other DNA or protein components. The enzyme is clever though, and will recombine only these sites, to generate attL and attR products; it will not recombine any other site combination. Nonetheless, phage Bxb1 encodes a second protein, gp47, that instructs gpInt to act on a different site pair - attL and attR - to mediate prophage excision. This represents an interesting molecular switch in protein function between two alternative choices of substrate, and is relevant to understanding other molecular switches in nature. The simplicity, directionality, and highly specific targeting, makes these recombination reactions highly suitable for adaptation to work in heterologous genetic systems including in other bacterial pathogens, malaria, in worms, fruit flies, mammalian cell culture, and in mice. Understanding and manipulating the Bxb1 system will therefore have a broad impact on the genetics of virtually all biological model organisms. PUBLIC HEALTH RELEVANCE: Elucidation of site-specific recombination mechanisms will facilitate the development of new vaccines for tuberculosis, powerful tools for manipulating the model organisms used to study human development, cancer, and infectious diseases such as malaria. Recombination is a core biological process and understanding its mechanisms and regulation will have a broad impact on our understanding of biology and medicine.